Substituted chroman derivatives, medicaments and use in therapy

ABSTRACT

Novel substituted chroman derivatives and intermediate compounds, compositions containing same, methods for their preparation and uses thereof as therapeutic agents particularly as anti-cancer and chemotherapeutic selective agents are described.

CROSS-REFERENCE

This application is a divisional of application Ser. No. 14/186,940,filed Feb. 21, 2014, which is continuation of application Ser. No.13/293,947, filed Nov. 10, 2011, now U.S. Pat. No. 8,697,891, which is acontinuation of application Ser. No. 12/551,277, filed Aug. 31, 2009,now U.S. Pat. No. 8,084,628, which is a divisional of application Ser.No. 11/230,726, filed Sep. 21, 2005, now U.S. Pat. No. 7,601,855, whichclaims benefit of Provisional Application No. 60/611,300, filed Sep. 21,2004, and Provisional Application No. 60/676,934, filed May 3, 2005, andclaims priority to International Application No. PCT/AU2004/001619,filed Nov. 19, 2004, the entire disclosures of said prior applicationsare hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to certain novel chroman derivatives andtheir salts and derivatives, compositions containing same, methods fortheir preparation and uses thereof as therapeutic agents particularly asanti-cancer and chemotherapeutic selective agents.

BACKGROUND OF THE INVENTION

Over 700 different naturally occurring isoflavones are known some ofwhich have biological properties with potential therapeutic benefit.

U.S. Pat. No. 5,726,202 generically discloses certain isoflavancompounds, particularly 3,4-diarylchroman and centchroman for thetreatment of benign prostatic hypertrophy.

WO 01/17986 also discloses certain isoflavan compounds.

SUMMARY OF THE INVENTION

Surprisingly, the present inventors have found a novel group ofcompounds of the general formula (I) which exhibit important therapeuticactivities including strong anti-cancer activity, chemotherapeuticselectivity and radiosensitisation of cancers.

Thus according to an aspect of the present invention there is provided acompound of the general formula (I):

-   or a salt or derivative thereof wherein:-   R₁ is hydrogen, hydroxy, halo, NR₁₀R₁₁, C₃₋₆ cycloalkyl, C₁₋₆    alkoxy, C₂₋₆ alkenyl, C₁₋₆ fluoroalkyl or C₁₋₆ alkyl optionally    substituted by one or more hydroxy, chloro, bromo, iodo or NR₁₀R₁₁    groups;-   the drawing “    ” and R₂ together represent a double bond or-   the drawing “    ” represents a single bond and R₂ is hydrogen, hydroxy, NR₁₀R₁₁,    C₁₋₃ alkoxy, C₁₋₃ fluoroalkyl, halo or C₁₋₃ alkyl optionally    substituted by one or more hydroxy, chloro, bromo, iodo or NR₁₀R₁₁    groups;-   R₃ is hydrogen, hydroxy, halo, NR₁₀R₁₁, C₃₋₆ cycloalkyl, C₁₋₆    alkoxy, C₁₋₆ fluoroalkyl, C₂₋₆ alkenyl, COOR₁₂, COR₁₃, (O)_(n)C₁₋₄    alkyleneNR₁₄R₁₅ or C₁₋₆ alkyl optionally substituted by one or more    hydroxy, chloro, bromo, iodo or NR₁₀R₁₁ groups;-   R₄, R₅, R₆, R₇, R₈ and R₉ are independently hydrogen, hydroxy, halo,    NR₁₀R₁₁, C₃₋₆ cycloalkyl, C₁₋₆ alkoxy, C₁₋₆ fluoroalkyl, C₂₋₆    alkenyl, COOR₁₂, COR₁₃ or C₁₋₆ alkyl optionally substituted by one    or more hydroxy, chloro, bromo, iodo or NR₁₀R₁₁;-   R₁₀, R₁₁ and R₁₂ are independently hydrogen, C₁₋₆ alkyl, C₃₋₆    cycloalkyl or trialkyl silyl;-   R₁₃ is hydrogen, C₁₋₆ alkyl, C₃₋₆ cycloalkyl or NR₁₀R₁₁;-   n represents 0 or 1; and-   R₁₄ and R₁₅ independently represent hydrogen or C₁₋₆ alkyl or    NR₁₄R₁₅ when taken together represents a 5 or 6 member    heteroaromatic or heterocylic;-   and pharmaceutically acceptable salts thereof,-   with the proviso that when R₁ represents hydrogen and “    ” is a single bond then R₂ does not represent hydrogen.

According to another aspect of the present invention there is provided aprocess for the preparation of a compound of formula (I).

According to another aspect of the present invention there is provided apharmaceutical composition which comprises one or more compounds offormula (I) or a pharmaceutically acceptable salt or derivative thereofin association with one or more pharmaceutical carriers, excipients,auxiliaries and/or diluents.

Thus, according to another aspect of the present invention there isprovided the use of a compound of formula (I) in therapy, particularlychemotherapy and as radiosensitising agents.

According to another aspect of the present invention there is provided amethod for the treatment, prevention or amelioration of a disease ordisorder, which comprises administering to a subject an effective amountof one or more compounds of the formula (I) or a pharmaceuticallyacceptable salt or derivative thereof optionally in association with acarrier and/or excipient.

According to another aspect of the present invention there is providedan agent for the treatment, prophylaxis or amelioration of a disease ordisorder which agent comprises one or more compounds of formula (I) or apharmaceutically acceptable salt or derivative thereof.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1. Shows pharmacokinetics and distribution of compound No. 1identified below in serum, faeces and urine. Average values arepresented for free and total concentrations of the compound (±SEM) as asemi-log plot in part A and as a standard linear plot in part B.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have found that compounds of the general formula(I) show surprising and unexpected biological and pharmaceuticalproperties.

The compounds of formula (I) of the invention are believed to havefavourable toxicity profiles with normal cells and good bioavailability.Surprisingly the compounds of the invention exhibit anti-canceractivity, significantly better than or at least comparable to knowncancer treatments.

The compounds of formula (I) are cytostatic and cytotoxic against abroad range of cancer cells of human and animal origin. By cancer cells,it is meant cells that display malignant characteristics and which aredistinguished from non-cancer cells by unregulated growth and behaviourwhich usually ultimately is life-threatening unless successfullytreated.

The cancer cells that have been found to be responsive to compounds offormula (I) are of epithelial origin (for example, prostate, ovarian,cervical, breast, gall-bladder, pancreatic, colorectal, renal, andnon-small lung cancer cells), of mesenchymal origin (for example,melanoma, mesothelioma and sarcoma cancer cells), and of neural origin(for example glioma cancer cells).

It is highly unusual and surprising to find a related group of compoundsthat display such potent cytotoxicity against cancer cells. Furthermoreit is thought that the compounds according to the invention also havelow toxicity against non-cancer cells such as keratinocytes derived fromhuman foreskin. Such cancer cell selectivity is highly unusual andunexpected.

Advantageously the compounds of formula (I) show cytotoxicity againstcancer cells that are well recognised for being poorly sensitive tostandard anti-cancer drugs. It is highly unusual and unexpected to findsuch potent activity against cancers, for example, cholangiocarcinoma,pancreatic adenocarcinoma and melanoma, that are highly resistant toknown anti-cancer drugs.

Advantageously the compounds of formula (I) also seem to display anability to radio-sensitise cancer cells, by which it is meant that thesecompounds either lower the amount of gamma-irradiation that is requiredto kill the cells, or they convert cancer cells from a state ofradio-resistance to radio-sensitivity.

Additionally the compounds of formula (I) are thought to possesschemo-sensitising activity, that is they increase the cytotoxicity ofchemotherapeutic agents, especially to cancer cells, and/or convertcancerous cells from a state of chemo-resistance to a chemo-sensitivestate.

Compounds of the invention may also provide chemo and/orradio-protective properties for non-cancerous cells. This hassignificant therapeutic implications because the traumatic side-effectsof chemotherapy and radiotherapy are caused by the toxicity of thetraditional treatments to non-cancerous cells.

The properties described above offer significant clinical advantages.

The radio and/or chemo-protective properties of the compounds of theinvention may be employed to protect healthy individuals from theeffects of radiation and/or chemical toxins, or lessen the effects ofthe same.

The properties described above offer significant clinical advantages.

The invention also provides the use of compounds of formula (I) to treatpatients with cancer by either reducing the rate of growth of suchtumours or by reducing the size of such tumours through therapy withsuch compounds alone, and/or in combination with each other, and/or incombination with other anti-cancer agents, and/or in combination withradiotherapy.

Generally in compounds of formula (I) according to the invention thesubstituents R₈ and R₉ will be distributed as shown below:

Generally in compounds of formula (I) according to the invention thesubstituents R₃, R₄

and R₅ will be distributed as shown below:

Preferably in compounds of formula (I) the drawing “

” represents a single bond.

Preferably in compounds of the invention, including compounds of formula(I) R₃ will be in the para-position.

In compounds of the invention, including compounds of formula (I) whenR₃ represents (O)_(n)C₁₋₄ alkyleneNR₁₄R₁₅ preferably it represents —OC₂alkyleneNR₁₄R₁₅ wherein NR₁₄R₁₅ represents pyrrolidinyl.

According to the invention there is provided compounds of formula (I-a):

or a salt or derivative thereof wherein

-   R₁, R₂, R₃, R₄, R₅, R₆, R₇ and R₈ are as defined above for compounds    of formula (I) with the proviso that when R₁ represents hydrogen and    “    ” is a single bond then R₂ does not represent hydrogen.

The positions of R₃, R₄ and R₅ shown above for compounds of formula (I)apply equally to compounds of formula (I-a).

The position of R₈ and R₉ shown above for compounds of formula (I)equally applies to compounds of formula (I-a).

In compounds of formula (I-a) R₇ preferably represents C₁₋₆ alkoxy orhydroxy, especially methoxy or hydroxy.

In compounds of formula (I-a) preferably “

” represents a single bond.

In another preferred aspect the invention there is provided compounds offormula (I-b):

or a salt or derivative thereof wherein

-   R₁ represents hydroxy, halo, NR₁₀R₁₁, C₃₋₆ cycloalkyl, C₋₆ alkoxy,    C₂₋₆ alkenyl, C₁₋₆ fluoroalkyl, C₁₋₆ alkyl optionally substituted by    one or more hydroxy, chloro, bromo, iodo or NR₁₀R₁₁ groups; and-   R₃, R₄, R₅, R₆, R₇, R₈ and R₉ are defined above for compounds of    formula (I).

The positions of R₃, R₄ and R₅ shown above for compounds of formula (I)apply equally to compounds of formula (I-b).

The position of R₈ and R₉ shown above for compounds of formula (I) applyequally to compounds of formula (I-b).

Preferably in compounds of formula (I-b) R₁ represents hydroxy, C₁₋₆alkoxy, C₁₋₆ fluoroalkyl, C₁₋₆ alkyl optionally substituted by one ormore hydroxy, chloro, bromo, iodo or NR₁₀R₁₁ groups, especially C₁₋₆alkyl, particularly methyl.

Preferably in compounds of formula (I-b) R₃ represents hydroxy, C₁₋₆alkoxy or C₁₋₆ alkyl optionally substituted by one or more hydroxy,chloro, bromo, iodo or NR₁₀R₁₁ groups, especially C₁₋₆ alkoxy orhydroxy, particularly methoxy.

Preferably in compounds of formula (I-b) R₃ is in the para position.

Preferably in compounds of formula (I-b) R₄, R₅ and R₆ independentlyrepresents hydrogen.

Preferably in compounds of formula (I-b) R₇ represents hydroxy or C₁₋₆alkoxy, especially hydroxy or methoxy.

Preferably in compound of formula (I-b) R₈ represents hydrogen, hydroxyor C₁₋₆ alkoxy, especially hydrogen, hydroxy or methoxy, particularlyhydrogen.

Preferably in compounds of formula (I-b) R₈ is in the 3 position.

Preferably R₉ in compounds of formula (I-b) represents hydrogen hydroxyor C₁₋₆ alkoxy, especially hydroxy or C₁₋₆ alkoxy, particularly hydroxyor methoxy.

Thus, in another aspect the invention provides compounds of the formula(I-bb):

or a salt or derivative thereof wherein

-   R₁, R₃, R₄, R₇, R₈ and R₉ are as defined above for compounds of    formula (I-b).

In a highly preferred embodiment, “

” represents a single bond.

The preferences expressed above for compounds of formula (I-b) applyequally to compounds of formula (I-bb).

Specific compounds within the scope of this first aspect of theinvention are as follows:

or a salt or derivative thereof.

Most preferably the compounds of formula (I-bb) have the followingstructure:

or salts or derivatives thereof.

In another preferred aspect the invention provides a compound of formula(I-c):

or a salt or derivative thereof wherein

-   R₂ represents hydroxy, halo, NR₁₀R₁₁, C₁₋₃ alkoxy, C₁₋₃ fluoroalkyl,    C₁₋₃ alkyl optionally substituted by one or more hydroxy, chloro,    bromo, iodo or NR₁₀R₁₁ groups; and R₃, R₄, R₅, R₆, R₇, R₈ and R₉ are    as defined above for compounds of formula (I).

The positions of R₃, R₄ and R₅ shown above for compounds of formula (I)apply equally to compounds of formula (I-b).

The position of R₈ and R₉ shown above for compounds of formula (I),where R₉ is in the para position apply equally to compounds of formula(I-b). NR₁₀R₁₁ in compounds of formula (I-c) preferably representshydrogen or C₁₋₃ alkyl, especially hydrogen or methyl.

Preferably in compounds of formula (I-c) R₂ represents hydroxy, methoxy,methyl or trifluoromethyl.

Preferably in compounds of formula (I-c) R₃ represents hydroxy, C₁₋₆alkoxy or C₁₋₆ alkyl optionally substituted by one or more hydroxy,chloro, bromo, iodo or NR₁₀R₁₁ groups, especially C₁₋₆ alkoxy, such asmethoxy, particularly methoxy. Preferably in compounds of formula (I-c)R₄, R₅ and R₆ independently represent hydrogen.

Preferably in compounds of formula (I-c) R₈ represents hydrogen, hydroxyor C₁₋₆ alkoxy, more preferably hydrogen or methoxy, especiallyhydrogen.

Preferably in compounds of formula (I-c) R₈ is situated in the 3position.

Preferably in compounds of formula (I-c) R₉ represents, hydrogen,hydroxy or C₁₋₆ alkoxy, especially hydroxy or C₁₋₆ alkoxy, particularlyhydroxy or methoxy.

More preferably in this second aspect the invention provides a compoundof formula (I-cc):

or a salt or a derivative thereof wherein:

-   R₂, R₃, R₄, R₆, R₇, R₈ and R₉ are defined above for compounds of    formula (I-c).

Preference expressed above for compounds of formula (I-c) apply equallyto compound of formula (I-cc).

Specific compounds of formula (I-cc) are shown below:

or a salt or a derivative thereof.

The compounds of formula (I) according to the invention include twochiral centres. The present invention includes all the enantiomers anddiastereoisomers as well as mixtures thereof in any proportions. Theinvention also extends to isolated enantiomers or pairs of enantiomers.Methods of separating enantiomers and diastereoisomers are well known toperson skilled in the art.

It will be clear to persons skilled in the art that in the compoundsaccording to the invention the aryl substituents on the heterocyclicring can be cis or trans relative to each other. Preferably in thecompounds according to the invention of formula (I) these substituentswill be cis.

A particularly preferred compound of the present invention is thecis-isomer of compound labelled compound No. 1 above.

Likewise, particularly preferred compounds are compound Nos. (2) to (9)in the cis-conformation.

Preferably, the salts of compounds according to the invention will bepharmaceutically acceptable salts.

The term alkyl is taken to include straight chain and branched chainsaturated alkyl groups of 1 to 6 carbon atoms, such as methyl, ethyl,propyl, isopropyl, butyl, isobutyl, sec-butyl, tertiary butyl, pentyland the like. The alkyl group more preferably contains preferably from 1to 4 carbon atoms, especially methyl, ethyl, propyl or isopropyl.

Cycloalkyl includes C₃₋₆ cycloalkyl such as cyclopropyl, cyclobutyl,cyclopentyl and cyclohexyl.

The alkyl group or cycloalkyl group may optionally be substituted by oneor more of fluorine, chlorine, bromine, iodine, carboxyl, C₁₋₄alkoxycarbonyl, C₁₋₄ alkylamino-carbonyl, di-(C₁₋₄alkyl)-amino-carbonyl, hydroxyl, C₁₋₄ alkoxy, formyloxy, C₁₋₄alkyl-carbonyloxy, C₁₋₄ alkylthio, C₃₋₆ cycloalkyl or phenyl.

Preferably the alkyl group does not bear any substituents. The term C₁₋₆alkoxy includes groups wherein the alkyl portion therein is a straightchain or branched chain alkyl moiety. C₁₋₆ alkoxy groups include:methoxy, ethoxy, propoxy, isopropoxy, butoxy, tertiary butoxy andsec-butoxy. Preferably the C₁₋₆ alkoxy substituents will be methoxy orethoxy, especially methoxy.

The term fluoroalkyl includes “alkyl” wherein one or more such as 1, 2,3, 4 or 5 of the hydrogens have been replaced by fluoro. The fluoroalkylmay be a straight chain or branched chain “alkyl” unit. Preferredfluoroalkyl groups include trifluoromethyl and pentafluoromethyl.

The term aryl is taken to include phenyl, benzyl, biphenyl and naphthyland may be optionally substituted by one or more C₁₋₄ alkyl, hydroxy,C₁₋₄ alkoxy, carbonyl, C₁₋₄ alkoxycarbonyl, C₁₋₄ alkylcarbonyloxy, nitroor halo.

The term “halo” is taken to include fluoro, chloro, bromo and iodo,preferably fluoro, chloro.

5 or 6 membered heterocyclic and heteroaromatic rings include: pyrrole,pyrroline, pyrrolidine, oxazoline, thiazole, imidazole, imidazoline,imidazolidine, pyrazole, pyrazoline, pyrazolidine, isoxazole,isothiazole, oxadiazole, furazan, triazole, thiadiazole, pyridine,piperidine, morpholine, thiomorpholine, pyridazine, pyrimidine,pyrazine, piperazine, triazine, thiadiazone and dithiazine each of whichmay be optionally substituted by one or more of fluorine, chlorine,bromine, iodine, carboxyl, C₁₋₄ alkoxycarbonyl, C₁₋₄alkylamino-carbonyl, di-(C₁₋₄ alkyl)-amino-carbonyl, hydroxyl, C₁₋₄alkoxy, formyloxy, C₁₋₄ alkyl-carbonyloxy, C₁₋₄ alkylthio or C₃₋₆cycloalkyl.

The compounds of the invention include all salts, such as acid additionsalts, anionic salts and zwitterionic salts, and in particular includepharmaceutically acceptable salts as would be known to those skilled inthe art. The term “pharmaceutically acceptable salt” refers to anorganic or inorganic moiety that carries a charge and that can beadministered in association with a pharmaceutical agent, for example, asa counter-cation or counter-anion in a salt. Pharmaceutically acceptablecations are known to those of skilled in the art, and include but arenot limited to sodium, potassium, calcium, zinc and quaternary amine.Pharmaceutically acceptable anions are known to those of skill in theart, and include but are not limited to chloride, acetate, tosylate,citrate, bicarbonate and carbonate.

Pharmaceutically acceptable salts include those formed from: acetic,ascorbic, aspartic, benzoic, benzenesulphonic, citric, cinnamic,ethanesulphonic, fumaric, glutamic, glutaric, gluconic, hydrochloric,hydrobromic, lactic, maleic, malic, methanesulphonic, naphthoic,hydroxynaphthoic, naphthalenesulphonic, naphthalenedisulphonic,naphthaleneacrylic, oleic, oxalic, oxaloacetic, phosphoric, pyruvic,para-toluenesulphonic, tartaric, trifluoroacetic, triphenylacetic,tricarballylic, salicylic, sulphuric, sulphamic, sulphanilic andsuccinic acid.

The term “pharmaceutically acceptable derivative” or “prodrug” refers toa derivative of the active compound that upon administration to therecipient is capable of providing directly or indirectly, the parentcompound or metabolite, or that exhibits activity itself and includesfor example phosphate derivatives and sulphonate derivatives. Thus,derivatives include solvates, pharmaceutically active esters, prodrugsor the like.

The preferred compounds of the present invention also include allderivatives with physiologically cleavable leaving groups that can becleaved in vivo to provide the compounds of the invention or theiractive moiety. The leaving groups may include acyl, phosphate, sulfate,sulfonate, and preferably are mono-, di- and per-acyl oxy-substitutedcompounds, where one or more of the pendant hydroxy groups are protectedby an acyl group, preferably an acetyl group. Typically acyloxysubstituted compounds of the invention are readily cleavable to thecorresponding hydroxy substituted compounds.

Chemical functional group protection, deprotection, synthons and othertechniques known to those skilled in the art may be used whereappropriate to aid in the synthesis of the compounds of the presentinvention, and their starting materials.

The protection of functional groups on the compounds and derivatives ofthe present invention can be carried out by well established methods inthe art, for example as described in T. W. Greene, Protective Groups inOrganic Synthesis, John Wiley & Sons, New York, 1981.

Hydroxyl protecting groups include but are not limited to carboxylicacid esters, eg acetate esters, aryl esters such as benzoate,acetals/ketals such as acetonide and benzylidene, ethers such asortho-benzyl and para-methoxy benzyl ether, tetrahydropyranyl ether andsilyl ethers such as tert-butyldimethyl silyl ether.

Protecting groups can be removed by, for example, acid or base catalysedhydrolysis or reduction, for example, hydrogenation. Silyl ethers mayrequire hydrogen fluoride or tetrabutylammonium fluoride to be cleaved.

It will be clear to persons skilled in the art of medicinal chemistrythat compounds of formula (I) may be converted into other compounds offormula (I), for example, where a compound of formula (I) bears one ormore hydroxyl substituents then one or more of these substituents can beconverted in to a halogen such as bromo, chloro or iodo by treating thealcohol with a halogenating agent, with use of protecting groups asrequired to protect other functionality in the molecule. Halogenatingagents include compounds like NBS, hydrobromic acid and chlorine gas.

Phenolic type hydroxyls may not be readily convertible to thecorresponding halogen compound by treatment with a halogenating agent.However, the desired halogen compound may be prepared by, for example,treating an appropriate aryl amine starting material with NaNO₂ in thepresence of HCl under reduced temperature conditions such as 0° C., toform the corresponding azide salt. Subsequent treatment with CuCl, CuBr,KI or HBF₄ may be used to convert the azide into the requiredhalo-compound.

A general process for preparing compounds of formula (I) comprises thesteps of:

i) treating a compound of formula (II):

or a protected derivative thereof wherein:

-   R₁, is hydrogen, hydroxy, NR₁₀R₁₁, C₃₋₆ cycloalkyl, C₁₋₆ alkoxy,    C₂₋₆ alkenyl, C₁₋₆ fluoroalkyl, C₁₋₆ alkyl optionally substituted by    one or more hydroxy or NR₁₀R₁₁ groups;-   R₆, R₇, R₈ and R₉ independently represent hydrogen, hydroxy,    NR₁₀R₁₁, C₃₋₆ cycloalkyl, C₁₋₆ alkoxy, C₁₋₆ fluoroalkyl, COOR₁₂,    COR₁₃ or C₁₋₆ alkyl optionally substituted by one or more hydroxy or    NR₁₀R₁₁ groups with a of formula (III):

or a protected derivative thereof wherein:

-   R₃ represents hydrogen, hydroxy, NR₁₀R₁₁, C₃₋₆ cycloalkyl, C₁₋₆    alkoxy, C₁₋₆ fluoroalkyl, C₂₋₆ alkenyl, COOR₁₂, COR₁₃, (O)_(n)C₁₋₄    alkyleneNR₁₄R₁₅, or-   C₁₋₆ alkyl optionally substituted by one or more hydroxy or NR₁₀R₁₁    groups;-   R₄ and R₅ independently represent hydrogen, hydroxy, NR₁₀R₁₁, C₃₋₆    cycloalkyl, C₁₋₆ alkoxy, C₁₋₆ fluoroalkyl, C₂₋₆ alkenyl, COOR₁₂,    COR₁₃ or C₁₋₆ alkyl optionally substituted by one or more hydroxy or    NR₁₀R₁₁ groups; and-   X represents a metallohalo moiety; and-   ii) optionally followed by converting the tertiary alcohol group on    the heterocyclic ring in the product formed to another substituent,    and-   iii) optionally followed by deprotection.

In step i) described above the compound of formula (III) is preferablyan organo metallic reagent which is reacted with the ketone compound offormula (II) under anhydrous conditions in an inert atmosphere such asunder nitrogen or argon, in an inert solvent such as THF(tetrahydrofuran), at a non-extreme temperature such as roomtemperature, or reduced temperature, for example, 0° C.

Suitable organometallic reagents include organolithium reagents,organomagnesium reagents and organocopper reagents. More preferably thearylating agent employed is an organomagnesium reagent such as aGringnard reagent, which may be prepared by reacting a compound offormula (III), wherein X represents halo such as bromo with magnesiummetal under anhydrous conditions in an inert atmosphere.

In step ii) described above the tertiary alcohol substituent on theheterocyclic ring in the product formed from the nucleophilic additionreaction may be converted into other R₂ substituents by known methods.For example, treatment with para-toluenesulfonic acid can be used toconvert the tertiary alcohol into a good leaving group. Thisintermediate tosylate may then be treated with a nucleophile such as ahydride source, an alcohol or an amine to provide the requiredsubstitution for the R₂ moiety.

Alternatively the tertiary hydroxyl may be converted to a halogen by useof a halogenating agent.

In a further aspect of the invention there is the dehydration of theproduct of said nucleophilic addition reaction to form a compound of thegeneral formula (I-d):

or a protected derivative thereof wherein R₁, R₃, R₄, R₅, R₆, R₇, R₈ andR₉ are as defined above for compounds of formula (I-b).

Dehydration can, for example, be catalysed by acid, by base orfacilitated by conversion of the tertiary alcohol into a better leavinggroup. Preferably compounds of formula (III) are dehydrated, forexample, by treatment with para-toluene sulphonic acid.

Preference expressed above for compounds of formula (I-b) apply equallyto compound of formula (I-d).

Specific compounds of formula (I-d) are shown below:

If required the double bond in the heterocycle in compounds of formula(I-d) can be removed by treatment with a reducing agent to provide othercompounds of formula (I). Reducing agents are well known to personsskilled in the art and can include hydride sources like borohydrides andalkali metal borohydrides, but would include hydrogen in catalytichydrogenation where a suitable catalyst such as palladium on carbon maybe used. Other suitable hydride sources include sodiumtriacetoxyborohydride tetrabutyl ammonium triacetoxyborohydride andsodium cyanoborohydride.

Preferably the double bond is reduced by hydrogenation.

Compounds of formula (II) may be prepared by reducing the double bond,preferably by hydrogenation, in the heterocyclic ring in compounds offormula (IV):

or a protected derivative thereof wherein:

-   R₁, R₆, R₇, R₈ and R₉ are as defined above for compound of formula    (II)

Access to compounds of general formula (IV) is available by generalsynthetic methods as set out in Scheme 1 below and as described inpublished International application No. WO01/17986, the disclosure ofwhich is incorporated herein by reference. The general synthetic methodis set out in Scheme 1.

Compounds for use in the preferred synthetic methods of the presentinvention may be derived from any number of sources readily identifiableto a person skilled in the art. For example, daidzein is readilyavailable or can be synthesised by standard methods known in the art.Suitable methods may be found in, for example, published InternationalPatent Applications WO 98/08503 and WO 00/49009, and references citedtherein, which are incorporated herein in their entirety by reference.

Clearly one or more of the above strategies may require the use of a oneor more protecting groups in order to protect functionality in otherparts of the molecule, when preforming a particular treatment or step.

Preferably any free alcohols, esters or other such reactive groups willbe protected, for example, as t-butyldimethylsilyl ethers duringnucleophilic addition reactions.

Chemical modifications and manipulations may be performed on thecompounds of the invention as would be known to one skilled in the art.For example, reaction of compound No. 1 with alkylating agents givesether derivatives at the free phenolic groups. Halogenation of thearomatic rings is also possible and, for example, reaction withN-bromosuccinimide affords the 8-bromo derivative (compound 42) as themain component, with smaller amounts of the 6-bromo isomer. Furtherreactions can include demethylation of alkoxy groups by employing, forexample, hydrogen bromide in acetic acid to afford the trihydroxycompound 43.

Additional compounds synthesised by the inventors include:

As used herein, the terms “treatment”, “prophylaxis” or “prevention”,“amelioration” and the like are to be considered in their broadestcontext. In particular, the term “treatment” does not necessarily implythat an animal is treated until total recovery. Accordingly, “treatment”includes amelioration of the symptoms or severity of a particularcondition or preventing or otherwise reducing the risk of developing aparticular condition.

The amount of one or more compounds according to the invention which isrequired in a therapeutic treatment will depend upon a number offactors, which include the specific application, the nature of theparticular compound used, the condition being treated, the mode ofadministration and the condition of the patient.

Compounds of formula (I) may be administered in a manner and amount asis conventionally practised. See, for example, Goodman and Gilman, “Thepharmacological basis of therapeutics”, 7th Edition, (1985). Thespecific dosage utilised will depend upon the condition being treated,the state of the subject, the route of administration and other wellknown factors as indicated above. In general, a daily dose per patientmay be in the range of 0.1 mg to 5 g; typically from 0.5 mg to 1 g;preferably from 50 mg to 200 mg. The length of dosing may range from asingle dose given once every day or two, to twice or thrice daily dosesgiven over the course of from a week to many months to many years asrequired, depending on the severity of the condition to be treated oralleviated.

It will be further understood that for any particular subject, specificdosage regimens should be adjust over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the compositions.

Relatively short-term treatments with the active compounds can be usedto cause stabilisation or shrinkage or remission of cancers. Longer-termtreatments can be employed to prevent the development of cancers inhigh-risk patients.

The production of pharmaceutical compositions for the treatment of thetherapeutic indications herein described are typically prepared byadmixture of the compounds of the invention (for convenience hereafterreferred to as the “active compounds”) with one or more pharmaceuticallyor veterinary acceptable carriers and/or excipients as are well known inthe art.

The carrier must, of course, be acceptable in the sense of beingcompatible with any other ingredients in the formulation and must not bedeleterious to the subject. The carrier or excipient may be a solid or aliquid, or both, and is preferably formulated with the compound as aunit-dose, for example, a tablet, which may contain up to 100% by weightof the active compound, preferably from 0.5% to 59% by weight of theactive compound.

The preferred concentration of active compound in the drug compositionwill depend on absorption, distribution, inactivation, and excretionrates of the drug as well as other factors known to those of skill inthe art. One or more active compounds may be incorporated in theformulations of the invention.

The formulations of the invention include those suitable for oral,rectal, ocular, buccal (for example, sublingual), parenteral (forexample, subcutaneous, intramuscular, intradermal, or intravenous),transdermal administration including mucosal administration via thenose, mouth, vagina or rectum, and as inhalants, although the mostsuitable route in any given case will depend on the nature and severityof the condition being treated and on the nature of the particularactive compound which is being used.

Formulation suitable for oral administration may be presented indiscrete units, such as capsules, sachets, lozenges, or tablets, eachcontaining a predetermined amount of the active compound; as a powder orgranules; as a solution or a suspension in an aqueous or non-aqueousliquid; or as an oil-in-water or water-in-oil emulsion. Suchformulations may be prepared by any suitable method of pharmacy whichincludes the step of bringing into association the active compound and asuitable carrier (which may contain one or more accessory ingredients asnoted above).

In general, the formulations of the invention are prepared by uniformlyand intimately admixing the active compound with a liquid or finelydivided solid carrier, or both, and then, if necessary, shaping theresulting mixture such as to form a unit dosage. For example, a tabletmay be prepared by compressing or moulding a powder or granulescontaining the active compound, optionally with one or more otheringredients.

Compressed tablets may be prepared by compressing, in a suitablemachine, the compound of the free-flowing, such as a powder or granulesoptionally mixed with a binder, lubricant, inert diluent, and/or surfaceactive/dispersing agent(s). Moulded tablets may be made by moulding, ina suitable machine, the powdered compound moistened with an inert liquidbinder.

Formulations suitable for buccal (sublingual) administration includelozenges comprising the active compound in a flavoured base, usuallysucrose and acacia or tragacanth; and pastilles comprising the compoundin an inert base such as gelatin and glycerin or sucrose and acacia.

Formulations suitable for ocular administration include liquids, gelsand creams comprising the active compound in an ocularly acceptablecarrier or diluent.

Compositions of the present invention suitable for parenteraladministration conveniently comprise sterile aqueous preparations of theactive compounds, which preparations are preferably isotonic with theblood of the intended recipient. These preparations are preferablyadministered intravenously, although administration may also be effectedby means of subcutaneous, intramuscular, or intradermal injection. Suchpreparations may conveniently be prepared by admixing the compound withwater or a glycine buffer and rendering the resulting solution sterileand isotonic with the blood. Injectable formulations according to theinvention generally contain from 0.1% to 60% w/v of active compound andcan be administered at a rate of 0.1 ml/minute/kg.

Formulations suitable for rectal administration are preferably presentedas unit dose suppositories. Formulations suitable for vaginaladministration are preferably presented as unit dose pessaries. Thesemay be prepared by admixing the active compound with one or moreconventional solid carriers, for example, cocoa butter, and then shapingthe resulting mixture.

Formulations or compositions suitable for topical administration to theskin preferably take the form of an ointment, cream, lotion, paste, gel,spray, aerosol, or oil. Carriers which may be used include Vaseline,lanoline, polyethylene glycols, alcohols, and combination of two or morethereof. The active compound is generally present at a concentration offrom 0.1% to 5% w/w, more particularly from 0.5% to 2% w/w. Examples ofsuch compositions include cosmetic skin creams.

Formulations suitable for transdermal administration may be presented asdiscrete patches adapted to remain in intimate contact with theepidermis of the recipient for a prolonged period of time. Such patchessuitably contain the active compound as an optionally buffered aqueoussolution of, for example, 0.1 M to 0.2 M concentration with respect tothe said active compound. See for example Brown, L., et al. (1998).

Formulations suitable for transdermal administration may also bedelivered by iontophoresis (see, for example, Panchagnula R, et al.,2000) and typically take the form of an optionally buffered aqueoussolution of the active compound. Suitable formulations comprise citrateor Bis/Tris buffer (pH 6) or ethanol/water and contain from 0.1 M to 0.2M active ingredient.

Formulations suitable for inhalation may be delivered as a spraycomposition in the form of a solution, suspension or emulsion. Theinhalation spray composition may further comprise a pharmaceuticallyacceptable propellant such as a hydrogen containing fluorocarbon such as1,1,1,2-tetrafluoroethane or 1,1,1,2,3,3,3-heptafluoro-n-propane.

The active compounds may be provided in the form of food stuffs, such asbeing added to, admixed into, coated, combined or otherwise added to afood stuff. The term food stuff is used in its widest possible sense andincludes liquid formulations such as drinks including dairy products andother foods, such as health bars, desserts, etc. Food formulationscontaining compounds of the invention can be readily prepared accordingto standard practices.

In a preferred aspect the invention provides a method of treating humansby administering an effective amount of one or more compounds accordingto the invention or a composition containing the same.

The active compound or pharmaceutically acceptable derivatives prodrugsor salts thereof can also be co-administered with other active materialsthat do not impair the desired action, or with materials that supplementthe desired action, such as antibiotics, antifungals,antiinflammatories, or antiviral compounds. The active agent cancomprise two or more isoflavones or derivatives thereof in combinationor synergistic mixture. The active compounds can also be administeredwith lipid lowering agents such as probucol and nicotinic acid; plateletaggregation inhibitors such as aspirin; antithrombotic agents such ascoumadin; calcium channel blockers such as verapamil, diltiazem, andnifedipine; angiotensin converting enzyme (ACE) inhibitors such ascaptopril and enalapril, and β-blockers such as propanolol, terbutalol,and labetalol. The compounds can also be administered in combinationwith nonsteriodal antiinflammatories such as ibuprofen, indomethacin,aspirin, fenoprofen, mefenamic acid, flufenamic acid and sulindac or anantiemetic such as Zofran®. The compounds can also be administered withcorticosteroids.

Compounds of formula (I) seem to be particularly suitable forco-administration with other anti-cancer drugs such as cisplatin and/ordehydroequol and/or paclitaxel (Taxol®). This may result in improvedeffects in the treatment in comparison to when only one of themedicaments is employed.

The co-administration may be simultaneous or sequential. Simultaneousadministration may be effected by the compounds being in the same unitdose, or in individual and discrete unit doses administered at the sameor similar time. Sequential administration may be in any order asrequired and typically will require an ongoing physiological effect ofthe first or initial active agent to be current when the second or lateractive agent is administered, especially where a cumulative orsynergistic effect is desired.

The invention also extends to novel intermediates employed in thepreparation of compounds according to the invention.

The compounds of the invention are useful in the treatment, preventionor amelioration of diseases associated with aberrant cell survival,aberrant cell proliferation, abnormal cellular migration, abnormalangiogenesis, abnormal estrogen/androgen balance, dysfunctional orabnormal steroid genesis, degeneration including degenerative changeswithin blood vessel walls, inflammation, and immunological imbalance.

The invention is further illustrated by the following non-limitingExamples and accompanying drawings.

EXAMPLES Example 13-(4-hydroxyphenyl)-4-(4-methoxyphenyl)-8-methyl-3,4-dihydro-2H-chromen-7-olStep 1 1-(2,4-Dihydroxy-3-methyl-phenyl)-2-(4-hydroxy-phenyl)-ethanone

2-Methylresorcinol (4.00 g, 1 equivalent) and 4-Hydroxyphenylacetic acid(5.00 g, 1 equivalent) were added to a round bottom flask. The roundbottom flask was attached to a condenser and placed in an oil bath, thewhole system was kept under nitrogen. Distilled BF₃.OEt₂ (20 ml, 5equiv.) was added to the mixture while stirring. The mixture wasrefluxed (110° C.). A yellow solid formed at 20 minutes indicating thatthe reaction had gone to completion. The reaction was left on heat for afurther 10 minutes and then cooled to room temperature. The yellow solidwas collected by suction filtration and washed with distilled water (200ml) to remove any excess BF₃.OEt₂ present. ¹H NMR in d-DMSO indicatedthe yellow solid was1-(2,4-Dihydroxy-3-methyl-phenyl)-2-(4-hydroxy-phenyl)-ethanone in >95%purity. The solid was dried on a freeze dryer for 24 hours (8.93 g,99%).

Step 2 7-Hydroxy-3-(4-hydroxy-phenyl)-8-methyl-chromen-4-one

1-(2,4-Dihydroxy-3-methyl-phenyl)-2-(4-hydroxy-phenyl)-ethanone (3.99 g)and N,N-DMF (115 ml) were added to a 500 ml 2-neck round bottom flask,the flask was attached to a condenser and placed in an oil bath. Adropping funnel was attached to the round bottom flask, and the wholesystem was kept under nitrogen. The flask was heated and maintained at50° C. BF₃.OEt₂ (57 ml, 29 equiv.) was added drop wise to the solutionover a period of 15 minutes, producing fumes. Methane sulfonyl chloride(MeSO₂Cl) (14 ml, 12 equiv.) was added to N,N-DMF (14 ml) in thedropping funnel. This mixture was then added drop wise to the roundbottom flask over a period of 10 minutes. Once addition was complete,the temperature was increased to reflux (110° C.). The reaction wasmonitored by HPLC (NV06_R&D.m) and was completed at 1 hr and 44 mins.The mixture was cooled to room temperature and poured into chilledstirred distilled water (4 L). A bright yellow floccular precipitate wasimmediately produced and the mixture was left stirring in the cold roomovernight. The mixture was then filtered through a buchner funnel, togive a yellow solid. ¹HNMR of the solid in d-DMSO indicated it was7-Hydroxy-3-(4-hydroxy-phenyl)-8-methyl-chromen-4-one with >95% purity.The solid was dried on a freeze dryer for 24 hours. When dry, the solidwas weighed (2.73 g, 66%).

Step 3 Acetic acid 3-(4-hydroxy-phenyl)-8-methyl-4-oxo-4H-chromen-7-ylester

Hydroxy-3-(4-hydroxy-phenyl)-8-methyl-chromen-4-one (35.18 g) werecombined into a round bottom flask (1 L). Pyridine (38 ml, 2equivalents) and acetic anhydride (576 ml, 47 equivalents) were added tothe round bottom flask while stirring at room temperature. The reactionwas monitored by HPLC and was completed instantaneously. There was achange in colour observed, the reaction mixture was dark brown initiallyand went bright orange with tan brown floccular particles upon stirring.The reaction mixture was poured into chilled, distilled H₂O (4 L) andwas left stirring at room temperature for 30 minutes. An off-white solidwas collected by suction filtration. ¹HNMR of the solid in d-CDCl₃indicated it was Acetic acid3-(4-hydroxy-phenyl)-8-methyl-4-oxo-4H-chromen-7-yl ester with >95%purity The solid was dried on a freeze dryer for 24 hours. When dry, thesolid was weighed (31.80 g, 69%).

Step 4 Acetic acid 3-(4-hydroxy-phenyl)-8-methyl-4-oxo-chroman-7-ylester

Acetic acid 3-(4-hydroxy-phenyl)-8-methyl-4-oxo-4H-chromen-7-yl ester(26.32 g), 10% Pd/Al₂O₃ (12.93 g, 50%) and ethyl acetate (EtOAc) (1.5 L)were added to a hydrogenation round bottom flask (2 L). The flask wasplaced on the hydrogenator, evacuated, purged with nitrogen gas (×5) andhydrogen gas (×5). The reaction was monitored by HPLC. 10% Pd/Al₂O₃ (9g, 35%) was added to the round bottom flask at 40 hours, indicated therewas no product peak present, only starting material was present. HPLC at62 hours indicated that the major peak was product peak with startingmaterial peak being half height of product peak. 10% Pd/Al₂O₃ (5.69 g,20%) was added to the round bottom flask to speed up reaction rate.Reaction was completed at 64 hours. The reaction mixture was filteredthrough celite to remove the Pd/Al₂O₃ catalyst, the celite was rinsedwith EtOAc (1 L) to ensure majority of product was collected. The EtOAcwas evaporated off on a rotary evaporator to give a yellow solid. Thesolid was re-crystallised in 95% EtOH (650 ml) and left in the freezerovernight. Off-white crystals were collected by suction filtration.¹HNMR in d-CDCl₃ indicated the off-white crystals were 8Acetic acid3-(4-hydroxy-phenyl)-8-methyl-4-oxo-chroman-7-yl ester with >95% purity.The crystals were stored in a desiccator for 24 hours, and weighed(18.37 g, 69%).

Step 5 7-Hydroxy-3-(4-hydroxy-phenyl)-8-methyl-chroman-4-one

Acetic acid 3-(4-hydroxy-phenyl)-8-methyl-4-oxo-chroman-7-yl ester(18.37 g), imidazole (21.18 g, 6 equivalents) and 100% EtOH (536 ml)were added to a round bottom flask (2 L). The reaction mixture wasrefluxed and monitored by HPLC. The reaction was completed at 8 hours.The reaction mixture was reduced (˜130 ml) on a rotary evaporator andpoured into stirred, chilled distilled water (1.9 L). The water crashout was left stirring in the cold room overnight. The pale pink solidwas collected by suction filtration. ¹H NMR of the solid indicated itwas 7-Hydroxy-3-(4-hydroxy-phenyl)-8-methyl-chroman-4-one with >95%purity. The solid was dried on the freeze dryer for 3 hours (8.31 g,59%).

Step 6 7,4′-Bis tert-butyldimethylsilyloxy-8-methyl-dihydrodaidzein

8-methyldihydrodaidzein 4.2 g, imidazole 13 g, tert-butyldimethylsilylchloride 12.7 g (70 mmoles), and N,N-DMF 50 ml were combined in a 250mIL round bottom flask and stirred under nitrogen at room temperaturefor 16 hours. The reaction was quenched with the addition of chilledwater (100 ml) with the reaction mix cooled in an ice bath. A whitesolid was filtered off, rinsed with water. Recrystallisation fromethanol afforded white fluffy crystals 3.2 g.

Step 77-(tert-butyldimethylsilyloxy)-3-3-(4-(tert-butyldimethylsilyloxy)phenyl-4-(4-methoxypenyl)-8-methyl-3,4-dihydro-2H-chromen-4-ol

2.5 g of the product of step 6 was weighed in a 2-neck round bottomflask, and flushed under nitrogen. Anhydrous THF 10 ml was added to thereaction vessel to give a clear slightly yellow solution. A condenserwas attached and the reaction vessel placed in an ice bath. Commercial4-methoxyphenylmagnesium bromide (0.5M solution in THF) 22.5 ml wasadded to the reaction mix dropwise over 10 minutes. The reaction wasquenched by the dropwise with wet ether (50:50 H₂O:diethyl ether) whilestill under nitrogen, with a white precipitate forming as increasingamounts of H₂O added. A further amount of water was added to thereaction mix before extraction with diethyl ether.

The organic layers were combined and washed with water, brine, driedover anhydrous magnesium sulphate and solvent removed in vacuo to giveclear yellow oil which solidified overnight to give an off-white solid.The oily nature of the material precluded an accurate yield beingcalculated. There was no further clean up of the product before use inthe next reaction. The oily nature of the material precluded an accurateyield being calculated.

Step 8 3-(4-hydroxyphenyl)-4-(4-methoxyphenyl)-2H-chromen-7-ol

4.2 g of the product of step 7, pTsOH (para-toluene sulphoric acid) 4.5g boiling chips and 200 ml of ethanol were combined in a 2-neck 500 mlround bottom flask with condenser attached. The reaction was heated atreflux for 3 hours. The solvent was concentrated in vacuo to ˜20 mlbefore being poured into chilled, stirred water (100 ml). The mixturewas then extracted with ethyl acetate, the combined organic layerswashed with water (3×100 ml), brine (1×100 ml), dried over anhydrousmagnesium sulphate and filtered and solvent removed in vacuo to givered/brown oil. The oil was dissolved in methanol (˜15 ml) and put infreezer overnight.

A white precipitate had formed overnight which was filtered off andrinsed with methanol. The filtrate was concentrated in vacuo to give abrown oil.

Step 93-(4-hydroxyphenyl)-4-(4-methoxyphenyl)-8-methyl-3,4-dihydro-2H-chromen-7-ol

2.5 g of the product of step 8, 10% Pd/Al₂O₃ 0.4 g and 50 ml of ethanolwere combined in a 2-neck 100 ml round bottom flask. The reaction washydrogenated at low pressure using standard conditions for 3 hours. Thereaction was filtered through Celite to remove the catalyst, rinsedthrough with ethanol (100 ml). The filtrate was concentrated to ˜15 mlbefore being poured into chilled, stirred water (300 mL). A pale orangeprecipitate formed which then formed a brown oil. The mixture was thenextracted with diethyl ether, the combined organic layers washed withwater (3×100 ml), brine (1×100 ml), dried over anhydrous magnesiumsulphate and filtered. The solvent was removed in vacuo to givered/brown oil. The product was recrystallised from diethyl ether (˜15ml), to give brown solid which was rinsed with chilled diethyl ether togive off-white crystals. 4 crops of (IV), ˜1 g. The ¹H NMR spectrum andnumbering scheme being shown below.

Peak H □ppm Shape J Hz Integrates Comments C2equatorial 4.13 m 1 C2axial4.25 m 1 C3 3.31 m 1 Partially obscured by water peak C4 4.28 m 1 C56.44 d 8.050 1 C6 6.30 d 8.4 1 C8-Me 2.0 s — 3 C2′, C6′ 7.5 d 8.7 2 C3′,C5′ 6.92 d 8.7 2 C2″, C6″ 6.60 d 8.7 2 C3″, C5″ 6.43 d 8.7 2 OMe 3.8 s 03

Example 23-(4-hydroxyphenyl)-4-(4-methoxyphenyl)-3′,4′-dimethoxy-8-methyl-3,4-dihydro-2H-chromen-7-olStep 1.1 1-(2,4-dihydroxy-3-methyl-phenyl)-2-(3,4-dimethoxy-phenyl)ethanone

2-Methylresorcinol (6.285 g, 1 equivalent) and 3,4-dimethoxyphenylaceticacid (9.251 g, 1 equivalent) were added to a round bottom flask. Theround bottom flask was attached to a condenser and placed in an oilbath, the whole system was kept under nitrogen. Distilled borontrifluoride diethyl etherate, BF₃.OEt₂ (42 ml, 5 equiv.) was added tothe mixture while stirring. The mixture was refluxed (110° C.). A yellowsolid formed at 75 minutes indicating that the reaction had gone tocompletion. The reaction was heated for a further 10 minutes and cooledto room temperature. The yellow solid was collected by suctionfiltration and washed with distilled water (200 ml) to remove any excessBF₃.OEt₂ present. ¹H NMR in d-DMSO indicated the yellow solid was1-(2,4-dihydroxy-3-methyl-phenyl)-2-(3,4-dimethoxy-phenyl) ethanonein >95% purity. The solid was dried on a freeze dryer for 24 hours(6.303 g, 43%).

Step 2.1 3-(3,4-dimethoxy-phenyl)-7-hydroxy-8-methyl-chromen-4-one

1-(2,4-dihydroxy-3-methyl-phenyl)-2-(3,4-dimethoxy-phenyl) ethanone(1078-1-49; 9.999 g, 34.4 mmol) was dissolved in N,N-DMF (15 mL), driedwith MgSO₄. Under an N₂ atmosphere, distilled BF₃—OEt₂ (16.08.04) wasadded dropwise at r.t. Heat begun after 20 min. After 1 h, methanesulfonyl chloride in DMF (8 mL in 20 mL) was slowly added at 50° C. Thereaction mixture was heated to reflux for 1.5 h. The dull yellowsolution was added to 1.2 L of cold, vigorously stirred water which wasleft at 4° C. overnight. The dull yellow solid was collected byfiltration, then placed in water to remove residual BF₃—OEt₂. Solid wascollected by filtration and dried using a freeze-dryer overnight. ¹H NMRin d-DMSO indicated the yellow solid was3-(3,4-dimethoxy-phenyl)-7-hydroxy-8-methyl-chromen-4-one in 90% purity(8.85 g, 82%).

Step 3.1 acetic acid3-(3,4-dimethoxy-phenyl)-8-methyl-4-oxo-4H-chromen-7-yl ester

3-(3,4-dimethoxy-phenyl)-7-hydroxy-8-methyl-chromen-4-one (9.82 g, 31mmol), acetic anhydride (62 ml) and pyridine (6.2 ml) were combined in around bottom flask and heated to reflux. The reaction was cooled to roomtemperature after 3 hours of heating, and a crystalline solid formed.The solid was filtered and rinsed with H₂O (1 L). ¹H NMR in d-CDCl₃indicated pale brown crystals was acetic acid3-(3,4-dimethoxy-phenyl)-8-methyl-4-oxo-4H-chromen-7-yl ester in 90%purity (7.214 g, 71%).

Step 4.1 Acetic acid3-(3,4-dimethoxy-phenyl)-8-methyl-4-oxo-chroman-7-yl ester

3-(3,4-dimethoxy-phenyl)-8-methyl-4-oxo-4H-chromen-7-yl ester (1.12 g, 3mmol), 10% Pd/Al₂O₃ (0.501 g, 45% w/w) and dry EtOAc (100 ml) wereplaced in a 2-neck round bottom flask and placed on the hydrogenator.After 4 hours, 1 major product was observed. The reaction was purged andthe catalyst filtered off through celite. The filtrate was reduced togive a white solid. ¹H NMR in d-CDCl₃ indicated the solid was Aceticacid 3-(3,4-dimethoxy-phenyl)-8-methyl-4-oxo-chroman-7-yl ester in 85%purity (1.1 g).

Step 5.1 3-(3,4-Dimethoxy-phenyl)-7-hydroxy-8-methyl-chroman-4-one

Acetic acid 3-(3,4-dimethoxy-phenyl)-8-methyl-4-oxo-chroman-7-yl ester(1.1 g, 3.2 mmol) and imidazole (3.2 g, 47 mmol) were refluxed in EtOH(100 ml). The reaction was complete after 90 minutes and allowed to coolto room temperature before pouring into stirred H₂O (800 ml). A finewhite precipitate was filtered off and ¹H NMR in d-CDCl₃ indicated thesolid was 3-(3,4-Dimethoxy-phenyl)-7-hydroxy-8-methyl-chroman-4-onein >95% purity (0.31 g, 30%).

Step 6.1 7,4′-Bistert-butyldimethylsilyloxy-3′,4′-dimethoxy-8-methyl-dihydrodaidzein

3′,4′Dimethoxy-8-methyldihydrodaidzein 2 g, imidazole 6.8 g, tertButyldimethylsilyl chloride 6.3 g, and N,N-DMF 50 ml were combined in a250 mIL round bottom flask and stirred under nitrogen at roomtemperature for 16 hours. The reaction was quenched with the addition ofchilled water (100 ml) with the reaction mix cooled in an ice bath. Awhite solid was filtered off, rinsed with water. Recrystallisation fromethanol afforded white fluffy crystals 2.2 g

Step 7.17-(tert-butyldimethylsilyloxy)-3-3-(4-(tert-butyldimethylsilyloxy)phenyl)-4-(4-methoxypenyl)-3′,4′dimethoxy-8-methyl-3,4-dihydro-2H-chromen-4-ol

The product of step 6.1 above 2 g was weighed in a 2-neck round bottomflask, and flushed under nitrogen. Anhydrous THF 10 ml was added to thereaction vessel to give a clear slightly yellow solution. A condenserwas attached and the reaction vessel placed in an ice bath. Commercial4-methoxyphenylmagnesium bromide (0.5M solution in THF) 22.5 ml wasadded to the reaction mix dropwise over 10 minutes. The reaction wasquenched by the dropwise with wet ether (50:50 H₂O:diethyl ether) whilestill under nitrogen, with a white precipitate forming as increasingamounts of H₂O added. A further amount of water was added to thereaction mix before extraction with diethyl ether. The organic layerswere combined and washed with water, brine, dried over anhydrousmagnesium sulphate and solvent removed in vacuo to give clear yellow oilwhich solidified overnight to give an off-white solid. The oily natureof the material precluded an accurate yield being calculated. There wasno further clean up of the product before use in the next reaction. Theoily nature of the material precluded an accurate yield beingcalculated.

Step 8.13-(4-hydroxyphenyl)-4-(4-methoxyphenyl)-3′,4′-dimethoxy-8-methyl-2H-chromen-7-ol

2 g of the product of step 7.1 pTsOH 4.5 g boiling chips and 100 ml ofethanol were combined in a 2-neck 500 ml round bottom flask withcondenser attached. The reaction was heated at reflux for 3 hours. Thesolvent was concentrated in vacuo to ˜10 ml before being poured intochilled, stirred water (100 ml). The mixture was then extracted withethyl acetate, the combined organic layers washed with water (3×100 ml),brine (1×100 ml), dried over anhydrous magnesium sulphate and filteredand solvent removed in vacuo to give red/brown oil. The oil wasdissolved in methanol (˜15 ml) and put in freezer overnight.

A white precipitate had formed overnight which was filtered off andrinsed with methanol. The filtrate was concentrated in vacuo to give abrown oil, which was crashed out into water to give a pale brown solid.

Step 9.13-(4-hydroxyphenyl)-4-(4-methoxyphenyl)-3′,4′-dimethoxy-8-methyl-3,4-dihydro-2H-chromen-7-ol

1 g of the product of step 8.1 10% Pd/Al₂O₃ 0.2 g and 25 ml of ethanolwere combined in a 2-neck 100 ml round bottom flask. The reaction washydrogenated at low pressure using standard conditions for 3 hours. Thereaction was filtered through Celite to remove the catalyst, rinsedthrough with ethanol (100 ml). The filtrate was concentrated to ˜5 mlbefore being poured into chilled, stirred water (100 mL). A pale orangeprecipitate formed which then formed a brown oil. The mixture was thenextracted with diethyl ether, the combined organic layers washed withwater (3×100 ml), brine (1×100 ml), dried over anhydrous magnesiumsulphate and filtered. The solvent was removed in vacuo to givered/brown oil. The product was recrystallised from diethyl ether (˜5ml), to give brown solid which was rinsed with chilled diethyl ether togive off-white crystals. 4 crops of (IV), ˜0.2 g. The ¹H NMR spectrumand numbering scheme shown below.

Peak H Chemical shift Shape J Hz Integrates Comments C2equatorial 4.05 m1 C2axial 4.25 m 1 C3 3.38 m 1 Partially obscured by water peak C4 4.45m 1 C5 6.80 d 8.4 1 C6 6.38 d 8.4 1 C8-Me 2.1  s — 3 C2′ 7.1  d 2.1 1C5′ 6.95 d 8.4 1 C6′ 6.98 dd 2.1, 8.4 1 C2″, C6″ 6.66 d 8.7 2 C3″, C5″6.33 d 8.7 2 OMe x3 3.8, 3.82, 3.83 s 0   9

¹H N.m.r. assignments are provided for the following compounds:

Cpd. 14

Peak H δ ppm Shape J Hz Integrates Comments C2 3.30 dd  3.293, 11.709 1dd is slightly under H₂O peak C2 4.05 dd  3.659, 10.612 1 C3 4.62 dd10.612, 12.075 1 dd is overlapped C5 6.45 d Under C3′, C5′ doublet C66.20 dd 2.561, 8.416 1 C8 6.23 d 2.196 1 Adjacent to O C2′, C6′ 6.70 d8.782 2 C3′, C5′ 6.45 d 8.782 2 Integrates for 3 in total including C5C2″, C6″ 7.11 m 2 Overlapping with 4″, 3″, 5″, cannot measure J C3″, C5″7.11 m 2 Overlapping with 4″, 2″, 6″ cannot measure J C4″ 7.11 m 1Overlapping with 3″, 5″, 4″ cannot measure JCpd. 30

Peak Inte- H δ ppm Shape J Hz grates C Comments C2 4.95 s 2 69 C3 — C4 —C4a — 116.5 C5 6.43 d 8.416 1 130.5 C6 6.30 dd 2.561, 8.416 1 108 C7 156C8 6.30 d 2.561 1 102 Adjacent to O C8a 158 C1′ C2′, C6′ 6.79 d 8.416 2128 C3′, C5′ 6.49 d 8.416 2 114.5 C4′ 155 Adjacent to O C1″ C2″, C6″7.04 m 2 130 C3″, C5″ 7.27 m 2 129 Overlapping with 4″, cannot measure JC4″ 7.27 m 1 126.5 Overlapping with 3″, 5″, cannot measure JCpd. 12

Inte- H δ ppm Peak Shape J Hz grates Comments C2equatorial 4.30 dd3.659, 10.612 1 C2axial 4.69 dd 10.978 1 dd is overlapped C3 3.44 dd3.293, 10.978 1 C5 6.83 d 8.782 1 C6 6.43 dd 2.561, 8.416  1 C8 6.47 d2.561 1 C2′, C6′ 6.83 d 8.782 2 C3′, C5′ 6.71 d 8.782 2 C2″, C6″ 7.05 d8.782 2 C3″, C5″ 6.76 d 8.782 2 Me A 3.75 s — 3 Methoxy peaks areinterchangeable Me B 3.79 s — 3 Me C 3.79 s — 3Cpd. 28

H δ ppm Peak Shape J Hz Integrates Comments C2 5.05 s — 2 C5 6.75 d8.416 1 C6 6.38 dd 2.561, 8.416 1 C8 6.50 d 2.651 1 C2′, C6′ 6.90 d8.782 2 C3′, C5′ 6.67 d 8.782 2 C2″, C6″ 7.02 d 8.782 2 C3″, C5″ 6.81 d8.416 2 Me A 3.74 s — 3 Methoxy peaks are inter- changeable Me B 3.79 s— 3 Me C 3.81 s — 3Cpd. 31

H δ ppm Peak Shape J Hz Integrates Comments C2 4.93 s 2 C5 6.28 d 2.1961 C6 6.24 dd 2.196, 8.416 1 C8 6.47 d 8.416 1 C9 3.63 s 3 C2′, C6′ 6.82d 8.416 2 Overlapping C3′, C5′ 6.51 d 8.416 2 with C2′, C6′ C2″ 6.57 1C4″ 6.81 1 C5″ 7.20 t 8.050 1 C6″ 6.63 1Cpd. 10

Peak Inte- H δ ppm Shape J Hz grates Comments C2 3.30 dd 1 under H₂Opeak C2 4.02 dd  4.391, 11.343 1 C3 4.60 dd 10.612, 12.075 1 dd isoverlapped C5 6.21 d 2.196 1 C6 6.18 dd 2.561, 8.416 1 C8 6.42 d 8.416 1Overlapping with C3′, C5′ C9 2.21 s 3 C2′, C6′ 6.72 d 8.782 2 C3′, C5′6.44 d 8.416 2 Overlapping with C8 C2″, C6″ 6.94 d 8.050 2 C3″, C5″ 7.01d 8.050 2Cpd. 11

H δ ppm Peak Shape J Hz Integrates Comments C2 3.30 dd  3.659, 11.709 1dd is slightly under H₂O peak, measured J from previous NMR C2 4.05 dd 3.659, 10.612 1 C3 4.62 dd 10.978, 11.709 1 dd is overlapped C5 6.47 d8.416 1 Overlapping with C3′, C5′ doublet C6 6.20 dd 2.561, 8.416 1Overlapping with C8 doublet C8 6.23 d 2.196 1 Overlapping with C6 dd C93.65 s 3 C2′, C6′ 6.72 d 8.416 2 C3′, C5′ 6.45 d 8.416 2 Integrates for3 in total including C5 C2″, C6″ 6.70 d 8.782 2 C3″, C5″ 7.03 d 8.782 2Cpd. 27

Peak H δ ppm Shape J Hz Integrates Comments C2 4.94 s 2 C5 6.48 d 8.4161 C6 6.25 dd 2.561, 8.416 1 C8 6.30 d 2.561 1 C9 3.65 s 3 C2′, C6′ 6.81d 8.416 2 C2′, C3′, C2″, C3″ C3′, C5′ 6.53 d 8.782 2 based on previousC2″, C6″ 6.86 d 8.782 2 ¹H NMR C3″, C5″ 6.97 d 8.782 2 assignment forsimilar compounds. this assignment.2.0. Materials and Methods2.1. Tissue Culture

The human pancreatic cancer cell line, HPAC (CRL-2119) was routinelycultured in 1:1 mixture DMEM (Sigma) plus Ham's F12 (Sigma) mediumcontaining HEPES (15 mM), insulin (0.002 mg/ml), transferrin (0.005mg/ml), hydrocortisone, (40 ng/ml), epidermal growth factor (10 ng/ml).The ovarian cancer cell lines; CP70 was obtained as a gift from Dr. GilMor (Yale University) and cultured in a 1:1 mixture DMEM plus Ham's F12medium, and SKOV-3 was purchased from ATCC and cultured in McCoys 5amedium. The breast cancer cell line MDA-MB-468 were cultured inLeibovitz's L-15 medium. The melanoma cell line MM200 was obtained as agift from Peter Hersey (University of Newcastle) and A2058 was obtainedas a gift from Dr Peter Parsons (QIMR). Both were cultured in DMEMmedium.

An cultures were supplemented with 10% FCS (CSL, Australia), penicillin(100 U/ml), streptomycin (100 mg/ml), L-glutamine (2 mM) and sodiumbicarbonate (1.2 g/L), and cultured at 37° C. in a humidified atmosphereof 5% CO₂. All cell lines were purchased from ATCC (Maryland, USA)except where noted.

The normal cell line NFF (neonatal foreskin fibroblasts) was a gift fromDr. Peter Parsons (Queensland Institute of Medical Research). RK (rabbitkidney) cells were obtained from Miller Whalley (Macquarie University).Both cell lines were cultured in RPMI supplemented with 10% FCS (CSL,Australia), penicillin (100 U/ml), streptomycin (100 mg/ml), L-glutamine(2 mM) and sodium bicarbonate (1.2 g/L), and cultured at 37° C. in ahumidified atmosphere of 5% CO₂

2.2. Proliferation Assays

IC50 values were determined for each cell line. Cells were seeded in96-well plates at an appropriate cell density as determined from growthkinetics analysis and cultured for 5 days in the absence and presence ofthe test compounds. Cell proliferation was assessed after the additionof 20 μl of 3-4,5 dimethylthiazol-2,5-diphenyl tetrazolium bromide (MTT,2.5 mg/ml in PBS, Sigma) for 3-4 hrs at 37° C. according tomanufacturer's instructions. IC50 values were calculated from semi-logplots of % of control proliferation on the y-axis against log dose onthe x-axis.

2.3. Compound No. 1 Pharmacokinetics—Oral

Compound No. 1 labelled above was prepared as homogenous suspensions in1% CMC (m:v, water). The formulation was delivered orally by gavage tofemale BALB/c mice at a dosage of 50 mg/kg. Three animals were allocatedto each timepoint (15 min, 30 min, 1 hr, 4 hr and 24 hr). At eachrespective timepoint, animals were euthanised by cervical dislocationand blood collected. The concentration of free compound was analysed bymass spectroscopy.

3.0. Results

3.1. Normal Cell Toxicity

Duplicate cutotoxicity assays against rabbit kidney cells demonstratedthat compound No. 1 has mild toxicity against these cells (Table 1).When compared to cisplatin and phenoxodiol another benchmark againstwhich potential anti-cancer drugs can be tested the degree of toxicityexhibited by compound No. 1 was greater than both the comparatorcompounds (2 μM compound No. 1 vs 9.9 μM cisplatin).

TABLE 1 Relative toxicity of compound No. 1 and cisplatin against rabbitkidney cells. Analogue (IC50 uM) Antineoplastic Tissue/cell Compound(IC50 uM) type Designation Phenoxodiol No. 1 Cisplatin Kidney RabbitKidney >150 >60 NT Fibroblast Neonatal >150 ~2 9.85 ± 5 ForeskinFibroblasts (Human, NFF)3.2. In Vitro Efficacy Against Cancer Cells

When compared the IC50 values of phenoxodiol and compound No. 1, werecompared, compound No. 1 demonstrated superior activity (˜2-10 foldgreater) against the ovarian cancer cell line (CP70), the AR negative,p53 Mt prostate cancer cell line (PC3), ER negative (p53 mt) breastcancer cell line (MDA-MB-468 respectively), p53 Mt Glioma (HTB-138), andp53 Mt small cell lung cancer (Table 2). Compound No. 1 exhibitedanti-cancer activity comparable to that of phenoxodiol against all othercell lines tested (Table 2) except HT-29 which is a colorectal cellline. Compound No. 1 was also equipotent against the melanoma cell lineMM200 (Table 2.1).

Thus, compound No. 1 shows good anticancer activity against a broadrange of cancer cell types, including ovarian, prostate, breast, glioma,pancreatic lung, colorectal and melanoma.

TABLE 2.1 Comparison of compound No. 1 and Phenoxodiol cytoxicityagainst cell lines representative of different malignancies IndicationCell line Phenoxodiol Compound No 1 Ovarian A2780  1.7 ± 0.61 NT CCP7011.1 ± 23.6 1.7 Prostate PC3 9.1 ± 8   1.7 ± 0.5 Breast MDA-MB-468 8.9 ±4.8  0.6 ± 0.01 Glioma HTB-138 20.4 ± 17.5 1.1 Pancreatic HPAC 56.6 ±16.8 NT Lung NCI-H23 8.3 ± 6.7 1.9 ± 0.1 Colorectal HT-29 52.5 ± 19.517.44 Melanoma MM200 6.2 ± 4.5  1.0 ± 0.01

A number of cancer cell lines were tested against analogues of compoundNo. 1 and their IC50 values were compared. The results show thatcompound No. 1 is the best across a broad range of cell lines, whilstcompound No. 20 also showed good results against ovarian, prostatebreast leukaemia and melanoma cell lines. Compound Nos. 15 and 17 alsoshowed good results against ovarian and melanoma cell lines respectively(Table 2.2).

TABLE 2.2 Comparison of compound Nos. 1, 10, 11, 12 and 14 to 20cytoxicity against cell lines representative of different malignanciesCompounds (IC50 uM) Indication Cell line Cpd. 14 Cpd. 30 Cpd. 11 Cpd. 15Cpd. 27 Cpd. 31 Ovarian CP70 61.3 18 52.7 ± 13  65 ± 1.9 22.4 ± 0.7 53.4Prostate PC3 74 ± 4   45 ± 4.9 >100 >100   28 ± 7.3   47 ± 8.4 BreastMDA-MB-468 >100 >100 NT NT NT NT Glioma HTB-138 >100 45.7 86.3 >100 33.794.6 Pancreatic HPAC >100   41 ± 5.3 >100 >100 27.6 ± 5.3 34.6 leukaemiaCCRF-CEM 90.2 66.6 95.4 81.8 16.6 51.5 ± 12  (ALL) NSC LungNCI-H460 >100 43.5 ± 9.2 >100 >100 31.6 ± 4.9 76.3 Colorectal HT-29 NTNT NT NT NT >100 Melanoma MM200 72.8 33 ± 3 60.4 ± 9.2 NT 14.6 27.2 ±4.6 Compounds (IC50 uM) Indication Cell line Cpd. 10 Cpd. 12 Cpd. 28Cpd. 32 Cpd. 1 Ovarian CP70 27.5 >100  62 ± 44 17.7 ± 5.9 1.9 ± 0.4Prostate PC3 37.5 ± 0.4 32.8 ± 4.7  49 ± 16   13 ± 0.6 1.6 ± 0.8 BreastMDA-MB-468 NT 63.5 ± 4.6 30.5 ± 4    9.2 ± 5.1   1 ± 0.6 Glioma HTB-13860.2 47.7 62.4 29 ± 2 1.16 Pancreatic HPAC 50 86.3 ± 67  50.7 ± 35  35.851.7 ± 8   leukaemia CCRF-CEM 40.7 >100 >100 15.4 1.6 ± 1.2 (ALL) NSCLung NCI-H460 39.7 NT 41.6 39.3 ± 7.7  1.2 ± 0.45 Colorectal HT-29 NT49.5 ± 32  29.4 ± 4.4 24.8 ± 3.9 15.7 ± 2.4  Melanoma MM200 NT >100  64± 74 12.6 ± 3.7 0.78 ± 0.233.3. Compound No. 1 Pharmacokinetics—Oral

Oral pharmacokinetics was determined in the female BALB/c mouse. A Cmaxof 27.3 μM free compound No. 1 was observed in the sera 15 minutes postadministrations. Compound No. 1 was rapidly eliminated from sera with aconcentration of 8.2 μM observed after 30 min and 2.4 μM after 1 hrmaking the half life of this agent around 30 minutes (FIG. 1 and Table3). The majority of compound No. 1 in sera was present in its conjugatedstate with the concentration of total compound No. 1 (free plusconjugate) achieved being ˜100 μM 15 min post administration (1:4;free:total). The ratio of free:total decreased over time (1:12 after 30min and 1:13 after 1 hr). Rapid appearance of compound No. 1 in urine athigh concentrations (815 μM) 15 min post administration providesevidence that compound No. 1 is absorbed from the gastro intestinaltract and excreted via the kidneys.

Compound No. 1 concentrations in urine peaked at 30 min (2640 μM) withlevels declining to ˜1500 μM and 545 μM at 1 and 4 hr respectively postadministration. The majority of said compound was present in it'sconjugated form with only ≦2% as the free entity was noted at anytime-point. A large proportion of said compound was also excreted infaeces, which was observed at least 1 hr post administration. However,it is not possible to confirm whether hepatic excretion is a mode ofremoval for this compound due to the fact that no liver samples wereassessed and because the observation of the compound in faeces may bedue to residual compound powder not absorbed from the gastro intestinaltract. It is interesting to note that almost 100% of the compoundrecovered from faeces was present in it's free form.

TABLE 3 Compound No. 1 pharmacokinetics and distribution in serum,faeces and urine. Data are representative of Average ± SEM. Compound No.1 Concentration Serum Serum Faeces Faeces Urine Urine Time Free Total %Free Total % Free Total % (days) (uM) (uM) Free (uM) (uM) Free (uM) (uM)Free 0.25 27.3 ± 23   103.4 ± 36.5 26.4 17.1 ± 7.2 815 ± 268 2.1 0.5 8.2± 6.5 77.2 ± 8   10.6 22.1 ± 1.5 2640 ± 1190 0.8 1 2.4 ± 1.6 31.4 ± 9.47.6 1055 ± 913 1067 ± 924 98.9 26.3 ± 2.2 1535 ± 158  1.7 4  0.3 ± 0.02 5.2 ± 0.8 5.8 1792 ± 625 1861 ± 630 96.3  1.1 ± 0.1 545 ± 203 0.2 240.05 ± 0.02 0.56 ± 0.5 8.9  800 ± 670  815 ± 680 98.2  0.1 ± 0.1 7.04 ±3.6  1.4

When compared to the oral pharmacokinetic data of phenoxodiol andcompound No. 1 would appear to have a similar half life, however 10-20fold higher free concentrations of compound No. 1 were achieved after 15and 30 min. Approximately 2-fold higher concentrations were observedafter 1 hr.

TABLE 4 Comparative oral pharmacokinetic data for Compound No. 1 andPhenoxodiol. Compound No. 1 (uM) Phenoxodiol (uM) Time Free Total FreeTotal 0.25 27.3 ± 23    103 ± 36.5 3.3 ± 0.13 511.5 ± 99   0.5 8.2 ± 6.577.2 ± 8   2.9 ± 0.05 357 ± 82 1 2.4 ± 1.6 31.3 ± 9.4  1.5 ± 0.11   387± 22.8 4 0.33 ± 0.02 5.2 ± 0.7 1.3 ± 0.07 117.6 ± 42   24 0.05 ± 0.020.56 ± 0.49 0.15 ± 0.04  0.13 ± 0.1 Dose* 13.8 mg/ml 4.6 mg/ml4.0. Effect on murine macrophages (RAW 264.7) stimulated with LPS

The mouse macrophage cell line RAW 264.7 was cultured in DMEMsupplemented with foetal calf serum (FCS), 2 mM glutamine and 50 U/mlpenicillin/streptomycin. Subconfluent cells were detached from the flaskby gentle scraping and 24-well plates seeded at 5×10⁵ cells per well andallowed to adhere for 1 hr. Cells were then treated either test compoundat a concentration of 10 μM (in 0.025% DMSO) or vehicle alone, andincubated for 1 hr. LPS 50 ng/ml (LPS—Sigma-Aldrich) was then added.After incubation for 16 hrs, culture media was collected and stored at−80° C. for ecosanoid measurements using enzyme immunometric assays(PGE₂ and TXB₂—Cayman Chemical).

TABLE 5 Percentage change in eicosanoid synthesis after incubating testcompound at 10 μM compared with incubation with vehicle alone. Positivevalues indicate enhanced synthesis; negative values indicate inhibitionof synthesis and consequently suggest anti-inflammatory activity.Compound PGE₂ TXB₂ 1 −53.8 −15.9 14 −23.6 −40.9 30 −60.7 −62.6 11 −51.0−53.2 15 −38.3 −58.4 27 −84.2 −86.1 31 −41.4 −48.3 10 −23.1 −7.1 12−68.3 −34.0 28 −85.1 −50.1 32 −71.1 −34.45.0 Conclusions

Compound No. 1 exhibits marked toxicity toward primary explants ofnon-transformed neonatal foreskin fibroblasts at concentrations lessthan cisplatin (IC50=2 μM compound No. 1 vs 9 μM cisplatinrespectively). However, relative mild toxicity was noted against rabbitkidney cells (Compound No. 1 IC50>60 μM). Efficacy studies demonstratethat compound No. 1 is active against cell lines representative ofmelanoma (MM200) and glioma (HTB-128). However, compound seemsparticularly active against cell lines representative of prostate (PC3),breast (MDA-MB-468) and lung cancer (NCIHH-H23) which traditionally havebeen cancers which are very difficult to treat.

Compound No. 1 was moderately active against the colorectal cell lineHT-29.

Pharmacokinetic analysis of compound No. 1 revealed that oraladministration of the drug yields markedly higher concentrations of thefree form of the drug when compared to similarly administeredphenoxidiol at 15 and 30 min post administration. Compound No. 1 andphenoxodiol each exhibit similar t1/2 (˜30 min).

Preliminary formulation studies of compound No. 1 reveal that themolecule has moderate to low solublity in 20% HPBCD (11.2 mg/ml).

The invention has been described herein, with reference to certainpreferred embodiments, in order to enable the reader to practice theinvention without undue experimentation. However, a person havingordinary skill in the art will readily recognise that many of thecomponents and parameters may be varied or modified to a certain extentwithout departing from the scope of the invention. Furthermore, titles,headings, or the like are provided to enhance the reader's comprehensionof this document, and should not be read as limiting the scope of thepresent invention.

The entire disclosures of all applications, patents and publications,cited herein, if any, are hereby incorporated by reference.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification individually or collectively, andany and all combinations of any two or more of said steps or features.

The reference to any prior art in this specification is not, and shouldnot be taken as, an acknowledgment or any form of suggestion that thatprior art forms part of the common general knowledge in the field ofendeavour.

Selected Reference Articles

Constantinou A I, Mehta R, Husband A. 2003 Phenoxodiol, a novelisoflavone derivative, inhibits dimethylbenz[a]anthracene (DMSA)-inducedmammary carcinogenesis in female Sprague-Dawley rats. Eur J Cancer. 39,1012-8.

Constantinou A I, Husband A. 2002 Phenoxodiol(2H-1-benzopyran-7-0,1,3-(4-hydroxyphenyl)), a novel isoflavonederivative, inhibits DNA topoisomerase II by stabilizing the cleavablecomplex. Anticancer Res. 22, 2581-5.

Gamble, J R., Xia, P., Hahn, C., Drew, J., Drogemuller, C., Carter, C.,Walker, C., Brown, D M., Vadas, M A. 2003 Phenoxodiol, a derivative ofplant flavanoids, shows potent anti-tumour and anti-angiogenicproperties. Nature Medicine. Submitted.

Hersey, P and Zhang, X. D. 2001 How melanoma cells evade Trail-inducedapoptosis. Nature reviews, Cancer, 1, 142-150.

Kamsteeg, M., Rutherford, T., Sapi, E., Hanczaruk, B., Shahabi, S.,Flick, M., Brown, D. M and Mor, G. 2003 Phenoxodiol—an isoflavoneanalogue—induces apoptosis in chemo-resistant ovarian cancer cells.Oncogene; 22, 2611-20.

The invention claimed is:
 1. A composition comprising a compound offormula (I-b):

or a pharmaceutically acceptable salt thereof wherein: R₁ is C₁₋₆ alkyl;the drawing “—” and R₂ together represent a double bond or the drawing“—” represents a single bond and R₂ is hydrogen or hydroxy, R₃, R₄ andR₅ are independently hydrogen, hydroxy, C₁₋₆ alkoxy, or C₁₋₆ alkyl; R₆is hydrogen; R₇ is C₁₋₆ alkoxy; and R₈ and R₉ are independentlyhydrogen, hydroxy, halo, C₁₋₆ alkoxy, or C₁₋₆ alkyl; wherein saidcomposition is optionally in association with one or more pharmaceuticalcarriers, excipients, auxiliaries and/or diluents.
 2. The composition ofclaim 1, wherein R¹ is methyl.
 3. The composition of claim 1, wherein R⁷is methoxy.
 4. The composition of claim 1, further comprising ananti-cancer agent.
 5. The composition of claim 4, wherein theanti-cancer agent is cisplatin, dehydroequol, or paclitaxel.
 6. Thecomposition of claim 5, wherein the anti-cancer agent is dehydroequol.